In at least nine inherited diseases, polyglutamine expansions cause neurodegeneration associated with protein misfolding and the formation of ubiquitin-conjugated aggregates. The focus of this renewal application continues to be the polyglutamine disorder, Spinocerebellar ataxia type 3, also known as Machado-Joseph disease. Although expanded polyglutamine causes disease, functional properties of the individual host proteins also contribute to pathogenesis. We will explore newly discovered properties of the polyglutamine disease protein, ataxin-3, that link its normal function to ubiquitin pathways. Aim 1 will define the ubiquitin-binding specificity of ataxin-3, testing the hypothesis that cooperative action of ubiquitin interacting motifs mediates poly-ubiquitin binding by ataxin-3. Aim 2 will seek to establish that ataxin-3 is a ubiquitin-specific protease, define its preferred substrates, and determine whether polyglutamine expansion alters protease activity. Aim 3 will use inducible cell lines and viral-mediated gene transfer in vivo to test the hypothesis that ataxin-3, through its ubiquitin-linked activities, functions in neuronal protein quality control to suppress polyglutamine aggregation and toxicity. And Aim 4 will determine whether CHIP (C-terminus of Hsp70-interacting protein), a protein with dual roles as a co-chaperone and ubiquitin ligase, modulates polyglutamine toxicity in mouse models of disease. Together, the proposed studies build toward our longterm objective of understanding disease pathogenesis in order to develop rational therapies. They should provide insight into the pathogenic mechanisms of SCA3/MJD specifically, and polyglutamine diseases generally. They may also shed light on the role of protein quality control, and particularly of de-ubiquitinating enzymes, in age-related neurodegenerative diseases characterized by protein misfolding and aggregation.